Optimizing chili production in drought stress: combining Zn-quantum dot biochar and proline for improved growth and yield

The reduction in crop productivity due to drought stress, is a major concern in agriculture. Drought stress usually disrupts photosynthesis by triggering oxidative stress and generating reactive oxygen species (ROS). The use of zinc-quantum dot biochar (ZQDB) and proline (Pro) can be effective techniques to overcome this issue. Biochar has the potential to improve the water use efficiency while proline can play an imperative role in minimization of adverse impacts of ROS Proline, functioning as an osmotic protector, efficiently mitigates the adverse effects of heavy metals on plants by maintaining cellular structure, scavenging free radicals, and ensuring the stability of cellular integrity. That’s why current study explored the impact of ZQDB and proline on chili growth under drought stress. Four treatments, i.e., control, 0.4%ZQDB, 0.1 mM Pro, and 0.4%ZQDB + Pro, were applied in 4 replications following the complete randomized design. Results exhibited that 0.4%ZQDB + Pro caused an increases in chili plant dry weight (29.28%), plant height (28.12%), fruit length (29.20%), fruit girth (59.81%), and fruit yield (55.78%) over control under drought stress. A significant increment in chlorophyll a (18.97%), chlorophyll b (49.02%), and total chlorophyll (26.67%), compared to control under drought stress, confirmed the effectiveness of 0.4%ZQDB + Pro. Furthermore, improvement in leaves N, P, and K concentration over control validated the efficacy of 0.4%ZQDB + Pro against drought stress. In conclusion, 0.4%ZQDB + Pro can mitigate drought stress in chili. More investigations are suggested to declare 0.4%ZQDB + Pro as promising amendment for mitigation of drought stress in other crops as well under changing climatic situations.

www.nature.com/scientificreports/processes.These adaptations include modifications in plant structure, adjustments in growth rates, alterations in tissue osmotic potential, and enhancement of antioxidant defenses 4 .To overcome this issue use of proline, biochar and zinc (Zn) quantum dots are becoming popular.
Biochar has porous structure that significantly boosts water retention in soil, ensuring a steady supply for plant absorption [5][6][7][8][9][10][11] .It also aids in preserving and enhancing nutrient availability and decreasing the impact of abiotic stress 9,10,[12][13][14] .Furthermore, better uptake of nutrients via biochar application also promotes the chlorophyll contents and improve gas exchange attributes under stress condition 14 .Research has also indicated that quantum dots (QDs) possess the capability to improve plant growth and alleviate the impacts of oxidative stress by regulating functioning of antioxidants 15 .Additionally, the ZnO QDs also promote the uptake and translocation of nutrients which in turn enhance plant biomass 8,15 .
Proline, a crucial amino acid for plants under stress from drought, extreme temperatures, and salinity, serves as an osmoprotectant, safeguarding against dehydration by accumulating in cells 16 .Its antioxidant properties shield against oxidative damage caused by stressors like drought.Proline role in stabilizing proteins and membranes maintains cellular integrity 17 , while its influence on cellular balance and gene expression underscores its significance in facilitating plant growth amid tough conditions 18,19 .
Chilies are renowned for their remarkable versatility, finding applications in culinary, medicinal, and agricultural domains owing to their abundant content of capsaicinoids, vitamins, carotenoids, and minerals 20 .Heir culinary significance transcends geographical boundaries, enriching global dishes while offering potential health benefits through their antioxidant properties 21 .Nevertheless, chili farming encounters significant hurdles, particularly from drought stress.Inadequate water supply during crucial growth stages detrimentally impacts plant development, resulting in smaller fruits, diminished yield, and increased vulnerability to diseases and pests.Prolonged drought exacerbates these challenges by impairing the plant's capacity to synthesize essential compounds such as capsaicinoids and vitamins, thereby compromising the quality and quantity of chili harvests 22 .
That's why current study aims to explore the potential of proline foliar and zinc-quantum dot biochar (ZQDB) on chili plants cultivated in drought stress.This study is covering the knowledge gap regarding the use of proline and ZQDB as combined treatment to alleviate the drought stress in chili.The novelty of the current study lies in the combined application of proline and ZQDB for mitigating drought stress.It is hypothesized that applying proline and ZQDB might be effect technique to mitigate the adverse effects of drought stress on chili plants, potentially enhancing their growth and productivity.

Experimental site
In 2022, experimental research was carried out in research area of ResearchSolution, situated in Multan, Punjab, Pakistan.The research site's geographical coordinates are 30°15′49″N and 71°30′35″E.The climatic data of the experiment is provided in Fig. 1.

Zn-quantum dots biochar (ZQDB)
For synthesis of zinc quantum dots (ZQD) standard protocol was adopted as described by 8 .For the production of biochar, cabbage waste obtained from the local market at coordinates 30°11′29.8"N71°28′48.8"Ewas collected.The collected waste underwent initial sun-drying before undergoing pyrolysis under partially aerobic conditions at a temperature of 325 ± 5 °C.The characteristic of pre-experimental biochar is given in Table 1.This mixture of biochar and ZQD (99:1) underwent stirring for 24 h to facilitate the binding of quantum dots with biochar.Following this period, the ZQDB mixture was subjected to multiple washes with ethanol to eliminate unbound quantum dots.Subsequently, the quantum dots biochar blend underwent drying in a vacuum oven at 60 °C for 24 h.

Collecting, sterilization, and sowing of seeds
The chili seeds utilized were obtained from a licensed seed dealer authorized by the Government of Punjab, Pakistan.Before sowing, 5% sodium hypochlorite solution was used for sterilization.Each pot containing 5 kg of soil (the physicochemical attribute of pre-experimental soil is provided in Table 1) was initially seeded with 15 seeds.Post-germination, a careful thinning process was executed, resulting in the retention of 4 seedlings per pot 23 .

Fertilizer application
The application included N, P, and K at a rate of 58: 25: 25 kg/acre (0.15: 0.06: 0.06 g/pot) to fulfill the macronutrient needs.Urea, single superphosphate, and potassium sulfate fertilizers were utilized to fulfill the requirement of N, P and K.

Drought
To simulate conditions of normal soil moisture (No Drought = 65% FC) and drought stress (35% FC), the trial involved manipulating soil moisture levels using moisture meter (YIERYI 4 in 1; Shenzhen, Guangdong Province, China), adhering to a methodology suggested by 24 .

Growth attributes data collection
Harvesting was done in mid of august.Soon after harvesting data was collected for total plant dry weight (g/ plant), plant height (cm), the number of primary branches per plant, fruit length (cm), fruit girth (cm), fruit yield (kg/plant), and chlorophyll content.For dry weight analytical weight balance was used.

Chlorophyll content
For chlorophyll assessment 0.5 g of freshly harvested leaf samples were grinded in a pestle mortar with 20 ml of 80% acetone.Afterward, the resulting mixture underwent centrifugation at 3000 rpm for 15 min and absorbance was taken at 645 and 663 nm 25 .

Antioxidant assays
To assess SOD activity, nitro blue tetrazolium (NBT) was used as per standard protocol.The absorbance reading was taken at 560 nm 26 .For CAT activity enzymatic breakdown of hydrogen peroxide (H 2 O 2 ) was assessed at 240 nm 27 .For APX activity, the reation among ascorbic acid and H 2 O 2 was observed at 290 nm wavelength 28 .The quantification of malondialdehyde (MDA) content was done using thiobarbituric acid method 29 .

Electrolyte leakage
Uniform leaf sections having weight one gram were placed in a test tube having 20 ml of deionized water.The test tubes were then kept at a consistent temperature of 25 °C for 24 h and solution electrical conductivity (EC1) was measured using a calibrated EC meter.Following this the test tubes were again heated at 120 °C for 20 min in water bath and secdnf electrical conductivity measurement (EC2) was recorded 32 .

Fruit harvest, dry weight, and nutrient analysis
In our study, we implemented a methodology involving randomly selecting three plants for each set of replicates, which were subsequently divided into leaves, stems, and roots.These segmented plant parts underwent drying in an oven at 70 ± 8 °C for two days to establish their dry weights and elemental concentrations.All analyses of nutrients were conducted based on the dry-weight measurements.

N, P, and K leaves
In this study, the determination of nitrogen content followed a modified micro-Kjeldahl method described in previous research 33 .Potassium content analysis utilized a flame photometer connected to a continuous-flow system, specifically employing the microflow automated continuous-flow analyzer III from Italy.Phosphorus content quantification at 420 nm was conducted using a spectrophotometer based on the yellow color method, following procedures outlined in earlier work 34 .

Statistical analysis
The data was analyzed using conventional statistical methods 35 .The application of a two-way ANOVA was conducted using OriginPro software.Subsequent paired comparisons, graph generation, and principal component analysis were performed using OriginPro software 36 .

Ethics approval and consent to participate
We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue.So, it is not applicable.Study protocol must comply with relevant institutional, national, and international guidelines and legislation.Our experiment follows the with relevant institutional, national, and international guidelines and legislation Vol.:(0123456789)

Results
Plant height, dry weight, and number of primary branches/plant Under no drought stress (DS), the addition of 0.4% zinc quantum dots biochar (ZQDB) treatment showed a 6.30% increase in plant height, while the proline (Pro) treatment resulted in an 11.79% increase in contrast to the control.The combined treatment of 0.4%ZQDB + Pro exhibited a 17.09% increase in plant height compared to control under no DS.Under DS, 0.4%ZQDB showed a 9.77%, Pro treatment 17.08%, and 0.4%ZQDB + Pro resulted in 28.12% increase in plant height over control (Fig. 2A).
In case of no DS, adding 0.4%ZQDB resulted in 7.36% increase in plant dry weight over the control.Treatment Pro caused 14.57% and 0.4%ZQDB + Pro showed 22.95% increase in plant dry weight than control under no DS.The application of 0.4%ZQDB, Pro and 0.4%ZQDB + Pro caused 8.60, 18.24 and 29.28% increase in plant dry weight compared to control respectively (Fig. 2B).

Convex hull and hierarchical cluster analysis
The control group, 0.4%ZQDB treatment, Pro treatment, and the combined 0.4%ZQDB + Pro treatment occupy their region on the plot, indicating differences in how these treatments affect the variables represented by PC 1 and PC 2. The combined treatment stands out as it forms a centralized cluster, suggesting a distinct effect that differs from individual treatments and the control group (Fig. 8A).
The convex hull analysis was conducted on the dataset based on PC 1 and PC 2, explaining 98.26% and 0.70% of the variation, respectively.The stress distribution indicated two distinct clusters: No Drought and Drought Stress.Convex Hull identified a clear separation between these clusters based on their scores in PC 1 and PC 2. The no drought cluster exhibited scores ranging from 0.02774 to 7.65504 in PC 1 and from − 0.30565 to 1.58993 in PC 2. Meanwhile, the drought stress cluster had scores ranging from − 7.08654 to − 0.17159 in PC 1 and from − 0.48373 to 0.77093 in PC 2. The Convex Hull method delineated the boundary encompassing these distinct groups, illustrating the pronounced separation between the samples experiencing no drought and those under drought stress based on their PC 1 and PC 2 scores (Fig. 8B).
The hierarchical cluster analysis was performed on variables, revealing distinct similarity linkages between various attributes.The analysis identified several clusters based on the similarity in their characteristics.Notably, the variables related to plant physiological traits formed control groups.For instance, Plant dry weight and total chlorophyll exhibited a similarity of 0.07143, indicating a close association between these parameters.Similarly, EL and SOD shared a similarity of 0.09983, suggesting a correlation in their responses.
Further, traits such as fruit length and chlorophyll b displayed a similarity of 0.16232, indicating a relationship between these attributes.Additionally, parameters like no. of primary branches/plant and fruit yield showed a similarity of 0.19166, hinting at a potential connection in their impact on plant productivity.Variables such as fruit girth and leave N exhibited a similarity of 0.30958, indicating a possible relationship between these traits.Chlorophyll a and leave K were similar to 0.21396 and 0.32587, respectively, pointing towards potential interdependencies between these physiological attributes.Interestingly, leave Na and leave P showed a similarity of 0.32657 and 0.54553, possibly suggesting distinct elemental responses within the plant system.The analysis also revealed strong associations within specific physiological attributes, such as H 2 O 2 and APX, displaying a high similarity of 0.66736, indicative of a close relationship in their responses.Moreover, the hierarchical clustering identified a distinct group comprising plant height, showing a significant similarity of 1.52116, implying a unique attribute set apart from the other variables analyzed (Fig. 8C).

Pearson correlation analysis
The correlation analysis revealed strong positive relationships among several plant traits.For instance, variables such as plant dry weight and total chlorophyll exhibited a notably high correlation of 0.99859, indicating a close positive association between these attributes.Similarly, attributes like no. of primary branches/plant and fruit yield displayed a strong positive correlation of 0.99621, suggesting a close relationship between the number of branches and fruit yield.Other variables, such as chlorophyll a and b, demonstrated a high positive correlation of 0.99323, indicating a closely linked behavior between these chlorophyll types.Additionally, fruit length and girth showed a strong positive correlation of 0.97541, signifying a relationship between fruit size characteristics.Conversely, specific attributes displayed strong negative correlations.For instance, EL and H 2 O 2 showcased a substantial negative correlation of − 0.99375, suggesting an inverse relationship between electrolyte leakage and www.nature.com/scientificreports/hydrogen peroxide levels.Similarly, leave N and leave P exhibited a negative correlation of − 0.99695, indicating a negative association between the levels of nitrogen and phosphorus in leaves (Fig. 9).

Drought stress
Drought-induced stress is a prominent abiotic factor impacting crops, initiating biochemical alterations.It significantly hinder plant growth, delay development, and decrease productivity 37 .The roots actively seek to absorb increased amounts of water as they expand, thereby enabling plants to adjust and reduce water loss through stomatal closure during periods of water scarcity 38 .Common signs of drought stress in plants comprise leaf curling, stunted growth, yellowing foliage, leaf burning, and irreversible wilting 39 .

Proline
Proline, an osmotic protector, facilitates plant growth under stress condition 40 .It not only act as an osmotolerant, but also act as a nutritional source i.e., K + , Ca + , P and N 41,42 .Furthermore, stabilization of mitochondrial electron transport complex, proteins, membranes and enzymes i.e., RUBISCO by exogenous application of proline are also allied factors which played an important role in enhancement of plant growth under stress conditions [43][44][45][46][47] .Additionally, proline has been observed to accumulate in actively dividing meristematic tissues, including the www.nature.com/scientificreports/root tip, shoot apex, lateral buds, inflorescence, and germinating seed.It serves as an energy source to sustain these metabolically demanding processes 48,49 .

Zinc quantum dots
It has been reported that ZnO quantum dots (QDs) facilitated the absorption and accumulation of essential nutrients i.e., Ca, Fe, Mg, Mn, B and Zn.Such improvement in nutrients, enhanced the soluble sugar that resulted in improvement of biomass and quality 15 .In combination with arbuscular mycorrhizae, application of ZQDB significantly enhanced the antioxidant activity i.e., POD, SOD and CAT 12 .POD is involved in scavenging harmful hydrogen peroxide (H 2 O 2 ) molecules, which are generated as byproducts of various metabolic processes.It helps to prevent the accumulation of reactive oxygen species (ROS), thereby reducing oxidative damage to cellular components 50 .SOD catalyzes the dismutation of superoxide radicals (O 2− ) into hydrogen peroxide (H 2 O 2 ) and molecular oxygen (O 2 ) thus alleviate oxidative stress and protects cells from oxidative damage 51 .CAT is another crucial antioxidant enzyme which catalyzes the decomposition of hydrogen peroxide into water and oxygen molecule 52 .

Biochar
Biochar serves as a valuable tool in alleviating osmotic stress within plants through several key mechanisms 53 .Firstly, its porous structure enables biochar to absorb and retain water, effectively increasing the soil's water holding capacity 54 .This feature becomes particularly beneficial during dry periods, as it ensures a more consistent moisture supply to plant roots, reducing the risk of osmotic stress.Secondly, biochar enhances soil structure by promoting aggregation and reducing compaction, which facilitates better water infiltration and root penetration 55 .Consequently, plants can access water more readily, mitigating the effects of osmotic stress.Moreover, biochar's high cation exchange capacity (CEC) enables it to adsorb and retain essential nutrients in the soil, ensuring their availability to plants even under stressful conditions 56 .This nutrient retention capability is crucial for maintaining plant health and resilience to environmental stressors like osmotic stress.Additionally, biochar stimulates microbial activity in the soil, fostering a healthy soil microbiome that enhances nutrient cycling and promotes shoot and root growth 57 .By supporting these beneficial soil microorganisms, biochar indirectly contributes to the mitigation of osmotic stress by improving nutrient uptake and overall plant vigor 6,14,58 .

Conclusion
In conclusion, use of 0.4% Zn-quantum dot biochar (ZQDB) with 0.1 mM proline is effective amendment for mitigating negative effects of drought stress.Specifically, 0.4% ZQDB + 0.1 mM Pro can reduce electrolyte leakage and increase the concentration of N, P and K, Such improvements in nutrients concentration and regulation of

Figure 2 .
Figure 2. Influence of ZQDB and proline on plant height (A), plant dry weight (B), and no. of primary branches/plant (C) of chili cultivated under no drought and drought stress.The bars represent the mean of four replicates with standard error.The Tukey test revealed significant changes at p < 0.05, shown by the different letters on the bars.

Figure 3 .
Figure 3. Influence of ZQDB and proline on fruit length (A), fruit girth (B), and fruit yield (C) of chili cultivated under no drought and drought stress.The bars represent the mean of four replicates with standard error.The Tukey test revealed significant changes at p < 0.05, shown by the different letters on the bars.

Figure 4 .
Figure 4. Influence of ZQDB and proline on chlorophyll a (A), chlorophyll b (B), total chlorophyll (C), and electrolyte leakage (EL) (D) of chili cultivated under no drought and drought stress.The bars represent the mean of four replicates with standard error.The Tukey test revealed significant changes at p < 0.05, shown by the different letters on the bars.

Figure 5 .
Figure 5. Influence of ZQDB and proline on hydrogen peroxide (H 2 O 2 ) (A), malondialdehyde (MDA) (B), superoxide dismutase (SOD) (C), and ascorbate peroxidase (APX) (D) of chili cultivated under no drought and drought stress.The bars represent the mean of four replicates with standard error.The Tukey test revealed significant changes at p < 0.05, shown by the different letters on the bars.

Figure 6 .
Figure 6.Influence of ZQDB and proline on total phenols (A), Catalase (B), and DPPH (C) of chili cultivated under no drought and drought stress.The bars represent the mean of four replicates with standard error.The Tukey test revealed significant changes at p < 0.05, shown by the different letters on the bars.

Figure 7 .
Figure 7. Influence of ZQDB and proline on leave N (A), leave P (B), Leave K (C), and leave Na (D) of chili cultivated under no drought and drought stress.The bars represent the mean of four replicates with standard error.The Tukey test revealed significant changes at p < 0.05, shown by the different letters on the bars.

Figure 8 .
Figure 8. Cluster plot convex hull for treatments (A), drought levels (B), and hierarchical cluster plot (C) for studied attributes.